New emerging roles of CD133 in cancer stem cell: Signaling pathway and miRNA regulation

Cancer stem cells (CSC) are rare immortal cells within a tumor that are able to initiate tumor progression, development, and resistance. Advances studies show that, like normal stem cells, CSCs can be both self-renewed and given rise to many cell types, therefore form tumors. A number of cell surface markers, such as CD44, CD24, and CD133 are frequently used to identify CSCs. CD133, a transmembrane glycoprotein, either alone or in collaboration with other markers, has been mainly considered to identify CSCs from different solid tumors. However, the exactness of CD133 as a cancer stem cell biomarker has not been approved yet. The clinical importance of CD133 is as a CSC marker in many cancers. Also, it contributes to shorter survival, tumor progression, and tumor recurrence. The expression of CD133 is controlled by many extracellular or intracellular factors, such as tumor microenvironment, epigenetic factors, signaling pathways, and miRNAs. In this study, it was attempted to determine: 1) CD133 function; 2) the role of CD133 in cancer; 3) CD133 regulation; 4) the therapeutic role of CD133 in cancers.     

Ablation of breast cancer stem cells with radiation

Tumor radioresistance leads to recurrence after radiation therapy. The radioresistant phenotype has been hypothesized to reside in the cancer stem cell (CSC) component of breast and other tumors and is considered to be an inherent property of CSC. In this study, we assessed the radiation resistance of breast CSCs using early passaged, patient-derived xenografts from two separate patients. We found a patient-derived tumor in which the CSC population was rapidly depleted 2 weeks after treatment with radiation, based on CD44(+) CD24(-) lin(-) phenotype and aldehyde dehydrogenase 1 immunofluorescence, suggesting sensitivity to radiotherapy. The reduction in CSCs according to phenotypic markers was accompanied by a decrease in functional CSC activity measured by tumor sphere frequency and the ability to form tumors in mice. In contrast, another patient tumor sample displayed enrichment of CSC after irradiation, signifying radioresistance, in agreement with others. CSC response to radiation did not correlate with the level of reactive oxygen species in CSC versus non-CSC. These findings demonstrate that not all breast tumor CSCs are radioresistant and suggest a mechanism for the observed variability in breast cancer local recurrence.     

CD166 promotes the cancer stem-like properties of primary epithelial ovarian cancer cells

Cancer stem cells (CSCs) or tumor-initiating cells are thought to play critical roles in tumorigenesis, metastasis, drug resistance, and tumor recurrence. For the diagnosis and targeted therapy of CSCs, the molecular identity of biomarkers or therapeutic targets for CSCs needs to be clarified. In this study, we identified CD166 as a novel marker expressed in the sphere-forming CSC population of A2780 epithelial ovarian cancer cells and primary ovarian cancer cells. The CD166⁺ cells isolated from A2780 cells and primary ovarian cancer cells highly expressed CSC markers, including ALDH1a1, OCT4, and SOX2, and ABC transporters, which are implicated in the drug resistance of CSCs. The CD166⁺ cells exhibited enhanced CSC-like properties, such as increased sphere-forming ability, cell migration and adhesion abilities, resistance to conventional anti-cancer drugs, and high tumorigenic potential in a xenograft mouse model. Knockdown of CD166 expression in the sphere-forming ovarian CSCs abrogated their CSC-like properties. Moreover, silencing of CD166 expression in the sphere-forming CSCs suppressed the phosphorylation of focal adhesion kinase, paxillin, and SRC. These results suggest that CD166 plays a key role in the regulation of CSC-like properties and focal adhesion kinase signaling in ovarian cancer.     

Cancer stem cells

Cancer stem cells (CSCs) are a small subpopulation of cells within tumors with capabilities of self-renewal, differentiation, and tumorigenicity when transplanted into an animal host. A number of cell surface markers such as CD44, CD24, and CD133 are often used to identify and enrich CSCs. A regulatory network consisting of microRNAs and Wnt/β-catenin, Notch, and Hedgehog signaling pathways controls CSC properties. The clinical relevance of CSCs has been strengthened by emerging evidence, demonstrating that CSCs are resistant to conventional chemotherapy and radiation treatment and that CSCs are very likely to be the origin of cancer metastasis. CSCs are believed to be an important target for novel anti-cancer drug discovery. Herein we summarize the current understanding of CSCs, with a focus on the role of miRNA and epithelial–mesenchymal transition (EMT), and discuss the clinical application of targeting CSCs for cancer treatment.     

Roles of Wnt/β-catenin signaling in the gastric cancer stem cells proliferation and salinomycin treatment

The Wnt1 protein, a secreted ligand that activates Wnt signaling pathways, contributes to the self-renewal of cancer stem cells (CSCs) and thus may be a major determinant of tumor progression and chemoresistance. In a series of gastric cancer specimens, we found strong correlations among Wnt1 expression, CD44 expression, and the grade of gastric cancer. Stable overexpression of Wnt1 increased AGS gastric cancer cells'' proliferation rate and spheroids formation, which expressed CSC surface markers Oct4 and CD44. Subcutaneous injection of nude mice with Wnt1-overexpressing AGS cells resulted in larger tumors than injection of control AGS cells. Salinomycin, an antitumor agent, significantly reduced the volume of tumor caused by Wnt1-overexpressing AGS cells in vivo. This is achieved by inhibiting the proliferation of CD44+Oct4+ CSC subpopulation, at least partly through the suppression of Wnt1 and β-catenin expression. Taken together, activation of Wnt1 signaling accelerates the proliferation of gastric CSCs, whereas salinomycin acts to inhibit gastric tumor growth by suppressing Wnt signaling in CSCs. These results suggest that Wnt signaling might have a critical role in the self-renewal of gastric CSCs, and salinomycin targeting Wnt signaling may have important clinical applications in gastric cancer therapy.     

A quantitative proteomics analysis of MCF7 breast cancer stem and progenitor cell populations

Accumulating evidence has demonstrated that breast cancers are initiated and develop from a small population of stem‐like cells termed cancer stem cells (CSCs). These cells are hypothesized to mediate tumor metastasis and contribute to therapeutic resistance. However, the molecular regulatory networks responsible for maintaining CSCs in an undifferentiated state have yet to be elucidated. In this study, we used CSC markers to isolate pure breast CSCs fractions (ALDH+ and CD44+CD24‐ cell populations) and the mature luminal cells (CD49f‐EpCAM+) from the MCF7 cell line. Proteomic analysis was performed on these samples and a total of 3304 proteins were identified. A label‐free quantitative method was applied to analyze differentially expressed proteins. Using the criteria of greater than twofold changes and p value <0.05, 305, 322 and 98 proteins were identified as significantly different in three pairwise comparisons of ALDH+ versus CD44+CD24‐, ALDH+ versus CD49f‐EpCAM+ and CD44+CD24‐ versus CD49f‐EpCAM+, respectively. Pathway analysis of differentially expressed proteins by Ingenuity Pathway Analysis (IPA) revealed potential molecular regulatory networks that may regulate CSCs. Selected differential proteins were validated by Western blot assay and immunohistochemical staining. The use of proteomics analysis may increase our understanding of the underlying molecular mechanisms of breast CSCs. This may be of importance in the future development of anti‐CSC therapeutics.     

Lung Cancer Stem Cells and Implications for Future Therapeutics

Lung cancer is the most dreaded of all cancers because of the higher mortality rates associated with it worldwide. The various subtypes of lung cancer respond differently to a particular treatment regime, which makes the therapeutic interventions all the more complicated. The concept of cancer stem cells (CSCs) is based primarily on the clinical and experimental observations that indicate the existence of a subpopulation of cells with the capacity to self-renew and differentiate as well as show increased resistance to radiation and chemotherapy. They are considered as the factors responsible for the cases of tumor relapse. The CSCs may have significant role in the development of lung tumorigenesis based on the identification of the CSCs which respond during injury. The properties of multi-potency and self-renewal are shared in common by the lung CSCs with the normal pluripotent stem cells which can be isolated using the similar markers. This review deals with the origin and characteristics of the lung cancer stem cells. The role of different markers used to isolate lung CSCs like CD44, ALDH (aldehyde dehydrogenase), CD133 and ABCG2 (ATP binding cassette sub family G member 2) have been discussed in detail. Analysis of the developmental signaling pathways such as Wnt/β-catenin, Notch, hedgehog in the regulation and maintenance of the lung CSCs have been done. Finally, before targeting the lung CSC biomarkers for potential therapeutics, challenges faced in lung cancer stem cell research need to be taken into account. With the accepted notion that the CSCs are to blame for cancer relapse and drug resistance, targeting them can be an important aspect of lung cancer therapy in the future.     

Hyaluronan constitutively regulates activation of COX-2-mediated cell survival activity in intestinal epithelial and colon carcinoma cells

Hyaluronan is a major component of the pericellular matrix surrounding tumor cells, including colon carcinomas. Elevated cycooxygenase-2 levels have been implicated in several malignant properties of colon cancer. We now show for the first time a strong link between hyaluronan-CD44 interaction and cyclooxygenase-2 in colon cancer cells. First, we have shown that increased expression of hyaluronan synthase-2 induces malignant cell properties, including increased proliferation, anchorage-independent growth, and epithelial-mesenchymal transition in HIEC6 cells. Second, constitutive hyaluronan-CD44 interaction stimulates a signaling pathway involving ErbB2, phosphoinositide 3-kinase/AKT, beta-catenin, and cyclooxygenase-2/prostaglandin E(2) in HCA7 colon carcinoma cells. Third, the HA/CD44-activated ErbB2 --> phosphoinositide 3-kinase/AKT --> beta-catenin pathway stimulates cell survival/cell proliferation through COX-2 induction in hyaluronan-overexpressing HIEC6 cells and in HCA7 cells. Fourth, perturbation of hyaluronan-CD44 interaction by hyaluronan oligomers or CD44-silencing RNA decreases cyclooxygenase-2 expression and enzyme activity, and inhibition of cyclooxygenase-2 decreases hyaluronan production suggesting the possibility of an amplifying positive feedback loop between hyaluronan and cyclooxygenase-2. We conclude that hyaluronan is an important endogenous regulator of colon cancer cell survival properties and that cyclooxygenase-2 is a major mediator of these hyaluronan-induced effects. Defining hyaluronan-dependent cyclooxygenase-2/prostaglandin E(2)-associated signaling pathways will provide a platform for developing novel therapeutic approaches for colon cancer.     

Loss of Tumor-Initiating Cell Activity in Cyclophosphamide-Treated Breast Xenografts

Cancer stem cells (CSCs) are a subpopulation of tumor cells with preferential tumor-initiating capacity and have been purported to be resistant to chemotherapy. It has been shown that breast CSC are, on average, enriched in patient tumors after combination neoadjuvant chemotherapy including docetaxel, doxorubicin, and cyclophosphamide (CPA). Here, we investigate the resistance of breast CSC to CPA alone in a xenograft model. CPA treatment led to a 48% reduction in tumor volume during a 2-week period. Cells bearing the CD44⁺ CD24^- phenotype were reduced by 90% (2.5% to 0.24%) in CPA-treated tumors, whereas cells with aldehyde dehydrogenase activity were reduced by 64% (4.7% to 1.7%). A subsequent functional analysis showed that CPA-treated tumors were impaired in their ability to form tumors, indicating loss of functional tumor-initiating activity. These results are consistent with a CSC phenotype that is sensitive to CPA and indicate that some patient CSC may not display the expected resistance to therapy. Deciphering the mechanism for this difference may lead to therapies to counteract resistance.     

Histone modifications: Targeting head and neck cancer stem cells

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and is responsible for a quarter of a million deaths annually. The survival rate for HNSCC patients is poor, showing only minor improvement in the last three decades. Despite new surgical techniques and chemotherapy protocols, tumor resistance to chemotherapy remains a significant challenge for HNSCC patients. Numerous mechanisms underlie chemoresistance, including genetic and epigenetic alterations in cancer cells that may be acquired during treatment and activation of mitogenic signaling pathways, such as nuclear factor kappa-light-chain-enhancer-of activated B cell, that cause reduced apoptosis. In addition to dysfunctional molecular signaling, emerging evidence reveals involvement of cancer stem cells (CSCs) in tumor development and in tumor resistance to chemotherapy and radiotherapy. These observations have sparked interest in understanding the mechanisms involved in the control of CSC function and fate. Post-translational modifications of histones dynamically influence gene expression independent of alterations to the DNA sequence. Recent findings from our group have shown that pharmacological induction of post-translational modifications of tumor histones dynamically modulates CSC plasticity. These findings suggest that a better understanding of the biology of CSCs in response to epigenetic switches and pharmacological inhibitors of histone function may directly translate to the development of a mechanism-based strategy to disrupt CSCs. In this review, we present and discuss current knowledge on epigenetic modifications of HNSCC and CSC response to DNA methylation and histone modifications. In addition, we discuss chromatin modifications and their role in tumor resistance to therapy.     

Tracing and targeting cancer stem cells: New venture for personalized molecular cancer therapy

Tumors consist of a mixture of heterogeneous cell types. Cancer stem cells(CSCs) are a minor sub-population within the bulk cancer fraction which has been foundto reconstitute and propagate the disease and to be frequently resistant to chemotherapy, irradiation, cytotoxic drugs and probably also against immune attack. CSCs are considered as the seeds of tumor recurrence, driving force of tumorigenesis and metastases. This underlines the urgent need for innovative methods to identify and target CSCs. However, the role and existence of CSCs in therapy resistance and cancer recurrence remains a topic of intense debate. The underlying biological properties of the tumor stem cells are extremely dependent on numerous signals, and the targeted inhibition of these stem cell signaling pathways is one of the promising approaches of the new antitumor therapy approaches. This perspective review article summarizes the novel methods of tracing CSCs and discusses the hallmarks of CSC identification influenced by the microenvironment or by having imperfect detection markers. In addition, explains the known molecular mechanisms of therapy resistance in CSCs as reliable and clinically predictive markers that could enable the use of new targeted antitumor therapy in the sense of personalized medicine.     

Cancer stem cells and angiogenesis

Cancer stem cells (CSCs) or tumor initiating cells were identified and characterized as a unique subpopulation with stem cell features in many types of cancer. Current CSC studies provide novel insights regarding tumor initiation, progression, angiogenesis, resistance to therapy and interplay with the tumor micro-environment. A cancer stem cell niche has been proposed based on these findings. The niche provides the soil for CSC self-renewal and maintenance, stimulating essential signaling pathways in CSCs and leading to secretion of factors that promote angiogenesis and long term growth of CSCs. We present evidence which has emerged over the past 5 years indicating interaction of CSCs with angiogenesis in the proposed "vascular niche". Based on these findings, targeting the "cancer stem cell niche" by combining an individualized anti-CSC approach with treatment of their microenvironment may represent a novel therapeutic strategy against solid tumor systems.     

Uncoupling Warburg effect and stemness in CD133<Superscript>+ve</Superscript> cancer stem cells from Saos-2 (osteosarcoma) cell line under hypoxia

Cancer stem cells (CSCs) which are known to be residing deep inside the core of the tumor in its hypoxia niche is responsible for relapse of cancers. Owing to this hypoxic niche, the residing CSCs simultaneously fuel their stemness, cancerous and drug resistance properties. Attributes of CSCs are still not properly understood in its hypoxia niche. Addressing this, we sorted CSCs from Saos-2 (osteosarcoma) cell line using CD133 antibody. The CD133^+ve CSCs exhibited quiescent cell proliferation in DNA doubling, Ca²⁺ signaling and cell cycle analysis. CD133^+ve CSCs exhibited increased production of ATP and lactate dehydrogenase (LDH) activity under hypoxia. CD133^+ve cells exhibited decreased glucose uptake compared to ATP levels under hypoxia. Moreover, there was only negligible LDH activity in CD133^+ve cells under normoxia which do not rely on Warburg effect. Stemness markers (such as c-Myc, SOX2, Oct4 and TERT), metastasis marker (CD44) and drug resistance marker (ABCG2) were highly expressed in CD133^+ve cells. In summary, both CD133^+ve/?ve cells of Saos-2 (osteosarcoma) cell line did not exhibit Warburg effect under normoxic condition. Moreover, this significantly indicates an uncoupling between stemness and Warburg effect in CD133^+ve. This work provides a novel insight into the metabolic and functional features of CSCs in a hypoxic environment which could open new avenues for therapeutic strategies aimed to target CSCs.     

CXCL2/CXCR2 axis induces cancer stem cell characteristics in CPT-11-resistant LoVo colon cancer cells via Gαi-2 and Gαq/11

Cancer stem cells (CSCs) exist in colon cancer and exhibit characteristics of stem cells which are due to lineages of tissues where they arise. Epithelial to mesenchymal transition (EMT)-undergoing cancer cells display CSC properties and therapeutic resistance. Cancer and stromal cells comprise of a tumor microenvironment. One way the two populations communicate with each other is to secret CXC ligands (CXCLs). CXCLs are capable of causing chemotaxis of specific types of stromal cells and control angiogenesis. Double immunofluorescence, western blot analysis, and colony-formation assay were carried out to compare parental and CPT-11-resistant LoVo cells. CPT-11-R LoVo colon cancer cells showed increased expression of CXCL1, CXCL2, CXCL3, and CXCL8. They displayed significantly increased intracellular protein levels of CXCL2 and CXCR2. CPT-11-R LoVo cells showed significantly elevated expression in aldehyde dehydrogenase 1 (ALDH1), cluster of differentiation 24 (CD24), cluster of differentiation 44 (CD44), and epithelial cell adhesion molecule (EpCAM). CXCL2 knockdown by short hairpin RNA resulted in reduced expression of CSC proteins, cyclins, EMT markers, G proteins, and matrix metalloproteinases (MMPs). Finally, Gαi-2 was found to promote expression of CSC genes and tumorigenesis which were more apparent in the resistant cells. In addition, Gαq/11 showed a similar pattern with exceptions of EpCAM and MMP9. Therefore, CXCL2–CXCR2 axis mediates through Gαi-2 and Gαq/11 to promote tumorigenesis and contributes to CSC properties of CPT-11-R LoVo cells.     

CD44 and CD24 cannot act as cancer stem cell markers in human lung adenocarcinoma cell line A549

Cancer stem cells (CSCs) are subpopulations of tumor cells that are responsible for tumor initiation, maintenance and metastasis. Recent studies suggested that lung cancer arises from CSCs. In this study, the expression of potential CSC markers in cell line A549 was evaluated. We applied flow cytometry to assess the expression of putative stem cell markers, including aldehyde dehydrogenase 1 (ALDH1), CD24, CD44, CD133 and ABCG2. Cells were then sorted according to the expression of CD44 and CD24 markers by fluorescence-activated cell sorting (FACS) Aria II and characterized using their clonogenic and sphere-forming capacity. A549 cells expressed the CSC markers CD44 and CD24 at 68.16% and 54.46%, respectively. The expression of the putative CSC marker ALDH1 was 4.20%, whereas the expression of ABCG2 and CD133 was 0.93%. Double-positive CD44/133 populations were rare. CD44⁺/24⁺ and CD44⁺/CD24^?/low subpopulations respectively exhibited 64% and 27.92% expression. The colony-forming potentials in the CD44⁺/CD24⁺ and CD44⁺/CD24^?/low subpopulations were 84.37 ± 2.86% and 90 ± 3.06%, respectively, while the parental A549 cells yielded 56.65 ± 2.33% using the colony-formation assay. Both isolated subpopulations formed spheres in serumfree medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). CD44 and CD24 cannot be considered potential markers for isolating lung CSCs in cell line A549, but further investigation using in vivo assays is required.     

Thoughts about cancer stem cells in solid tumors

Cancer chemotherapy efficacy is frequently impaired by either intrinsic or acquired tumor resistance.A fundamental problem in cancer research is identifying the cell type that is capable of sustaining neoplastic growth and its origin from normal tissue cells.In recent years,the cancer stem cell(CSC) theory has changed the classical view of tumor growth and therefore the therapeutic perspective.Overcoming intrinsic and acquired resistance of cancer stem/progenitor cells to current clinical treatments represents a major challenge in treating and curing the most aggressive and metastatic cancers.On the other hand,the identification of CSCs in vivo and in vitro relies on specific surface markers that should allow the sorting cancer cells into phenotypically distinct subpopulations.In the present review,recent papers published on CSCs in solid tumors(breast,prostate,brain and melanoma) are discussed,highlighting critical points such as the choice of markers to sort CSCs and mouse models to demonstrate that CSCs are able to replicate the original tumor.A discussion of the possible role of aldehyde dehydrogenase and CXCR6 biomarkers as signaling molecules in CSCs and normal stem cells is also discussed.The author believes that efforts have to be made to investigate the functional and biological properties of putative CSCs in cancer.Developing diagnostic/prognostic tools to follow cancer development is also a challenge.In this connection it would be useful to develop a multidisciplinary approach combining mathematics,physics and biology which merges experimental approaches and theory.Biological models alone are probably unable to resolve the problem completely.     

Cancer cells stemness: A doorstep to targeted therapy

Recent advances in research on cancer have led to understand the pathogenesis of cancer and development of new anticancer drugs. Despite of these advancements, many tumors have been found to recur, undergo metastasis and develop resistance to therapy. Accumulated evidences suggest that small population of cancer cells known as cancer stem cells (CSC) are responsible for reconstitution and propagation of the disease. CSCs possess the ability to self-renew, differentiate and proliferate like normal stem cells. CSCs also appear to have resistance to anti-cancer therapies and subsequent relapse. The underlying stemness properties of the CSCs are reliant on multiple molecular targets such as signaling pathways, cell surface molecules, tumor microenvironment, apoptotic pathways, microRNA, stem cell differentiation, and drug resistance markers. Thus an effective therapeutic strategy relies on targeting CSCs to overcome the possible tumor relapse and chemoresistance. The targeted inhibition of these stem cell biomarkers is one of the promising approaches to eliminate cancer stemness. This review article summarizes possible targets of cancer cell stemness for the complete treatment of cancer.